We describe the receptor binding and antagonistic properties of two novel nonpeptide antagonists, FR167344 (3-bromo-8-[2,6-dichloro-3-[N-[(E)-4-(N,N-dimethylcarbamoyl)cinnamido acetyl]-N-methylamino]benzyloxy]-2-methylimidazo[1,2-a]pyridine hydrochloride) and FR173657 (8-[3-[N-[(E)-3-(6-acetamidopyridin-3-yl)acryloylglycyl]-N-m ethylamino]-2,6-dichlorobenzyloxy]-2-methylquinoline), for the human bradykinin receptor subtypes (B1 and B2). In competitive experiments using membranes prepared from Chinese hamster ovary cells expressing the bradykinin receptor subtypes, FR167344 and FR173657 showed a high affinity binding to the B2 receptor with IC50 values of 65 and 8.9 nM, respectively, and no binding affinity for the B1 receptor. FR167344 and FR173657 inhibited the B2 receptor-mediated phosphatidylinositol (PI) hydrolysis and produced a concentration-dependent rightward shift in the dose-response curve to bradykinin. This shift was accompanied by a progressive reduction of maximal response. Estimated pA2 values for the antagonism of bradykinin-induced PI hydrolysis by FR167344 and FR173657 were 8.0 and 9.0, respectively. FR167344 and FR173657 showed no stimulatory effects on PI hydrolysis. Therefore, FR167344 and FR173657 are potent, highly selective, and insurmountable antagonists for the human bradykinin B2 receptor.

Kinins, members of a family of peptides released from kininogens by the action of kallikreins, exhibit a variety of biological activities including vasodilation, increased vascular permeability, contraction of smooth muscle cells, and activation of sensory neurons. However, investigation of the physiological actions of kinins has been greatly hampered because its effects are curtailed by rapid proteolysis in blood, lung, and liver. We describe the pharmacological characteristics of a novel nonpeptide bradykinin receptor agonist FR190997 (8-[2,6-dichloro-3-[N-[(E)-4-(N-methylcarbamoyl)cinnamidoacetyl ]-N-methylamino]benzyloxy]-2-methyl-4-(2-pyridylmethoxy)quinoli ne). FR190997 markedly stimulated phosphatidylinositol hydrolysis in Chinese hamster ovary cells permanently expressing the human bradykinin B2 receptor. The response of phosphatidylinositol hydrolysis was antagonized by the B2 receptor selective antagonist Hoe 140 (D-Arg-[hydroxyproline3,beta-thienylalanine4,D-Tic7,++ +Oic8]bradykinin). In competitive experiments using membranes prepared from Chinese hamster ovary cells expressing the human bradykinin receptor subtypes, FR190997 showed a high affinity binding to the B2 receptor with IC50 value of 5.3 nM and no binding affinity for the B1 receptor. In vivo, FR190997 mimics the biological action of bradykinin and induces hypotensive responses in rats with prolonged duration. Therefore, FR190997 is a highly potent and subtype-selective nonpeptide agonist which displays high intrinsic activity. This compound should represent a powerful tool for further investigation of the physiology and pathophysiology of bradykinin receptors.

Background. Refractoriness of glioblastoma multiforme (GBM) largely depends on its radioresistance. We investigated the radiosensitizing effects of celecoxib on GBM cell lines under both normoxic and hypoxic conditions. Methods. Two human GBM cell lines, U87MG and U251MG, and a mouse GBM cell line, GL261, were treated with celecoxib or g-irradiation either alone or in combination under normoxic and hypoxic conditions. Radiosensitizing effects were analyzed by clonogenic survival assays and cell growth assays and by assessing apoptosis and autophagy. Expression of apoptosis-, autophagy-, and endoplasmic reticulum (ER) stressrelated genes was analyzed by immunoblotting. Results. Celecoxib significantly enhanced the radiosensitivity of GBM cells under both normoxic and hypoxic conditions. In addition, combined treatment with celecoxib and g-irradiation induced marked autophagy, particularly in hypoxic cells. The mechanism underlying the radiosensitizing effect of celecoxib was determined to be ER stress loading on GBM cells. Conclusion. Celecoxib enhances the radiosensitivity of GBM cells by a mechanism that is different from cyclooxygenase-2 inhibition. Our results indicate that celecoxib may be a promising radiosensitizing drug for clinical use in patients with GBM. Keywords:autophagy, celecoxib, ER stress, glioblastoma, hypoxia, radiosensitivity. G lioblastoma multiforme (GBM) is the most frequently occurring malignant tumor of the central nervous system. 1 Despite standard carecomprising maximum surgical resection, 60 Gy of conventional radiotherapy, and chemotherapy with temozolomide (TMZ), the median survival time among patients with GBM is 1 year. 2The molecular mechanisms underlying the initiation and progression of GBM reported to date fall into 2 categories: (i) primary GBM with increased epidermal growth factor receptor (EGFR) expression and mutation of the phosphatase and tensin homolog and (ii) secondary GBM that is transformed from benign glioma types with stepwise mutations in the p53 and retinoblastoma genes. It is known that these genetic alterations in GBM are significantly associated with clinical prognosis.1 It has also been reported that methylation of O6-methylguanine-DNA methyltransferase increases the susceptibility of GBM tumor cells to alkylating agents, such as TMZ, 3 and mutation in the isocitrate dehydrogenase 1 gene was recently reported to significantly affect the prognosis in patients with GBM. 4 On the basis of these findings, therapy that targets these specific molecules could be a promising approach for controlling GBM. One possible target is cyclooxygenase-2 (COX-2) because a positive association has been reported among the levels of COX-2, EGFR/EGFRvIII, and activated signal transducer and activator of transcription 3 (STAT3).5 COX-2 expression was reportedly increased in human GBM and was negatively correlated with clinical outcomes in patients. 6 Celecoxib, a nonsteroidal anti-inflammatory drug (NSAID), selectively inhibits This drug is used clinically both for patients with ...

One-third of patients with medulloblastoma die due to recurrence after various treatments including radiotherapy. Although it has been postulated that cancer stem-like cells are radio-resistant and play an important role in tumor recurrence, the “stemness” of medulloblastoma cells surviving irradiation has not yet been elucidated. Using a medulloblastoma cell line ONS-76, cells that survived gamma irradiation were investigated on their “stemness” in vitro. From 10 500 cells, 20 radio-resistant clones were selected after gamma ray irradiation (5 Gy × two fractions) using the replica micro-well technique. These 20 resistant clones were screened for CD133 positivity by flow cytometry followed by side population assay, tumor sphere formation assay and clonogenic survival assay. Results revealed CD133 fractions were significantly elevated in three clones, which also exhibited significantly increased levels of tumor sphere formation ability and side population fraction. Clonogenic survival assay demonstrated that their radio-resistance was significantly higher than the parental ONS-76. This may support the hypothesis that a small number of cancer stem-like cells (CSCs) are the main culprits in local recurrence after radiotherapy, and disruption of the resistance mechanism of these CSCs is a critical future issue in improving the outcome of patients with medulloblastoma.

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